The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21&rsquo;s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.

fig6s2: Pol κ prevents the accumulation of DNA replication stress in p21 depleted cells independently of the siRNA used and in cells stably lacking p21.(A) U2OS cells were transfected with sip21 and 2 different siRNA for Pol κ. Western Blot analysis was performed to detect endogenous p21 and GFP- Pol κ. (B) RT-PCR was performed to detect mRNA levels of p21 and Pol κ using the indicated siRNAs. (C) IdU track length was measured using 2 different siRNAs for Pol κ in siLuc and sip21 depleted cells. 100fibers/sample were counted in 2 independent experiments. (D) Cells with more than five 53BP1 foci were analyzed in cells depleted of p21 and using 2 different siRNAs for Pol κ. (E) RT-PCR was performed in HCT116 p21+/+ and p21−/− cells to determine mRNA levels of Pol κ. (F) IdU track length in HCT116 p21+/+ and p21−/− depleted of Pol κ.100fibers/sample were counted in 2 independent experiments.DOI:http://dx.doi.org/10.7554/eLife.18020.015

Mentions:
T. Huang and colleagues have previously reported that MN accumulation induced by USP1 depletion depends on Pol κ (Jones et al., 2012). Therefore, we set to explore the effect of p21 on Pol κ recruitment to DNA replication factories. First, we observed that Pol κ foci were formed only in a modest percentage of control cycling cells (siLuc in Figure 5A–B). However, when p21 was depleted, the percentage of cells with Pol κ foci raised significantly (Figure 5A–B). Second, the interaction of PCNA and GFP-Pol κ in the chromatin fraction increased when p21 was depleted (Figure 5C). Third, using PLA an increase in the number of endogenous PCNA/Pol κ interacting foci was revealed in p21-depleted samples (Figure 5D–E). We hypothesized that an increased recruitment of Pol κ to the replication forks in p21-depleted cell may slow down DNA elongation, triggering fork collapse and/or the generation of under-replicated DNA. To test this hypothesis, Pol κ was down-regulated in p21-depleted cells (Figure 6A) and different DNA replication parameters were tested. Forty eight hours after siRNA transfection, Pol κ depletion alone had no effect on most parameters, except from a modest increase in RPA foci formation (Figure 6—figure supplement 1). Such result may be in agreement with the role of Pol κ in the replication of non-B DNA regions such as G4 cuadruplex (Betous et al., 2009). Notably however, the simultaneous elimination of Pol κ and p21 prevented all the phenotypes associated with p21 depletion. Specifically, Pol κ depletion rescued the defective nascent DNA elongation (Figure 6B), the origin frequency (Figure 6C), the percentage of EdU positive cells (Figure 6—figure supplement 1A) and the increased number of cells with chromatin bound-PCNA (Figure 6—figure supplement 1B). Similarly, markers of replication stress such as RPA foci (Figure 6—figure supplement 1C), γH2AX (Figure 6—figure supplement 1D) and 53BP1 foci (Figure 6D) were downregulated after simultaneous depletion of p21 and Pol κ. Similar results were obtained when using a second siRNA specific for Pol κ in U2OS cells (Figure 6—figure supplement 2A–D) and when employing a different cell line, HCT116 p21 −/− cells (Figure 6—figure supplement 2E–F).10.7554/eLife.18020.012Figure 5.The recruitment of pol κ to replication-associated structures increases in the absence of p21.

fig6s2: Pol κ prevents the accumulation of DNA replication stress in p21 depleted cells independently of the siRNA used and in cells stably lacking p21.(A) U2OS cells were transfected with sip21 and 2 different siRNA for Pol κ. Western Blot analysis was performed to detect endogenous p21 and GFP- Pol κ. (B) RT-PCR was performed to detect mRNA levels of p21 and Pol κ using the indicated siRNAs. (C) IdU track length was measured using 2 different siRNAs for Pol κ in siLuc and sip21 depleted cells. 100fibers/sample were counted in 2 independent experiments. (D) Cells with more than five 53BP1 foci were analyzed in cells depleted of p21 and using 2 different siRNAs for Pol κ. (E) RT-PCR was performed in HCT116 p21+/+ and p21−/− cells to determine mRNA levels of Pol κ. (F) IdU track length in HCT116 p21+/+ and p21−/− depleted of Pol κ.100fibers/sample were counted in 2 independent experiments.DOI:http://dx.doi.org/10.7554/eLife.18020.015

Mentions:
T. Huang and colleagues have previously reported that MN accumulation induced by USP1 depletion depends on Pol κ (Jones et al., 2012). Therefore, we set to explore the effect of p21 on Pol κ recruitment to DNA replication factories. First, we observed that Pol κ foci were formed only in a modest percentage of control cycling cells (siLuc in Figure 5A–B). However, when p21 was depleted, the percentage of cells with Pol κ foci raised significantly (Figure 5A–B). Second, the interaction of PCNA and GFP-Pol κ in the chromatin fraction increased when p21 was depleted (Figure 5C). Third, using PLA an increase in the number of endogenous PCNA/Pol κ interacting foci was revealed in p21-depleted samples (Figure 5D–E). We hypothesized that an increased recruitment of Pol κ to the replication forks in p21-depleted cell may slow down DNA elongation, triggering fork collapse and/or the generation of under-replicated DNA. To test this hypothesis, Pol κ was down-regulated in p21-depleted cells (Figure 6A) and different DNA replication parameters were tested. Forty eight hours after siRNA transfection, Pol κ depletion alone had no effect on most parameters, except from a modest increase in RPA foci formation (Figure 6—figure supplement 1). Such result may be in agreement with the role of Pol κ in the replication of non-B DNA regions such as G4 cuadruplex (Betous et al., 2009). Notably however, the simultaneous elimination of Pol κ and p21 prevented all the phenotypes associated with p21 depletion. Specifically, Pol κ depletion rescued the defective nascent DNA elongation (Figure 6B), the origin frequency (Figure 6C), the percentage of EdU positive cells (Figure 6—figure supplement 1A) and the increased number of cells with chromatin bound-PCNA (Figure 6—figure supplement 1B). Similarly, markers of replication stress such as RPA foci (Figure 6—figure supplement 1C), γH2AX (Figure 6—figure supplement 1D) and 53BP1 foci (Figure 6D) were downregulated after simultaneous depletion of p21 and Pol κ. Similar results were obtained when using a second siRNA specific for Pol κ in U2OS cells (Figure 6—figure supplement 2A–D) and when employing a different cell line, HCT116 p21 −/− cells (Figure 6—figure supplement 2E–F).10.7554/eLife.18020.012Figure 5.The recruitment of pol κ to replication-associated structures increases in the absence of p21.

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21&rsquo;s PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.